Identification of a Novel Intronic Mutation in VMA21 Associated with a Classical Form of X-Linked Myopathy with Autophagy

Introduction   VMA21 -related myopathy is one of the rare forms of slowly progressive myopathy observed in males. Till now, there have been only a handful of reports, mainly from Europe and America, and two reports from India. Method  Here, we describe a case of genetically confirmed VMA21 -associated myopathy with clinical, histopathological, and imaging features with a list of known VMA21 mutations. Results  A 29-year-old man had the onset of symptoms at 18 years of age with features of proximal lower limb weakness. Muscle magnetic resonance imaging showed the preferential involvement of vasti and adductor magnus. A biopsy of the left quadriceps femoris showed features of autophagic vacuolar myopathy with vacuoles containing granular eosinophilic materials. In targeted next-generation sequencing, hemizygous mutation in the 3′ splice site of intron 2 of the VMA21 gene (c.164–7 T > A) was identified and confirmed the diagnosis of X-linked myopathy with excessive autophagy. Conclusion  This report expands the phenotypic and genotypic profile of VMA21 -related myopathy, with a yet unreported mutation in India.


Introduction
X-linked myopathy with excessive autophagy (XMEA) is one of the early-onset myopathies that predominantly affects males. 1 It results mainly from a genetic defect in VMA21 leading to alteration in chaperone assembly of vacuolar ATPase and increases the lysosomal pH, 2 hence also called with respiratory failure requiring ventilatory support. 5here are fewer than 50 cases reported worldwide.Here, we present the clinical, radiological, histopathological, and genetic features of a young man with XMEA from India.

Case History
A 29-year-old man born from nonconsanguineous parents was evaluated in 2019.He presented with gradually progressive weakness of lower limbs from 18 years of age.Initially, he noticed difficulty while cycling and running fast.Over the next year, he started experiencing buckling episodes and falls while walking.By 25 years of age, he developed difficulty rising from the floor and climbing stairs.There was no feature of distal limb weakness, myalgia, cramps, contractures, or cardiac symptoms.There was no significant positive family history (►Fig. 1).
On examination, he has mild calf hypertrophy.The muscle power, according to modified Medical Research Council grading, was as follows: infraspinatus (4), Iliopsoas (4), hip adductors (4 þ ), quadriceps (4), and the rest of the power examination was normal.Tendon reflexes were normal except for hypoactive knee jerks.There was no scapular winging or muscle contractures.The creatine kinase level was 6630 IU/L (normal: 20-200 U/L).The electrocardiogram and two-dimensional echocardiography were normal.Nerve conduction study was normal.
Muscle magnetic resonance imaging (MRI) showed bilateral symmetric volume loss with fatty replacement of the proximal thigh muscles, preferentially involving the vasti and adductor magnus with relative sparing of the rectus femoris.In the thigh muscles, there was no aberrant short tau inversion recovery signal intensity (►Fig. 2).
Open muscle biopsy was done from the left quadriceps femoris muscle and analyzed using hematoxylin and eosin and Masson's trichrome stains.Histopathology showed fiber size variation with scattered atrophic angulated fibers and a few hypertrophic fibers, along with internalized nuclei and splitting.A few of the fibers also showed vacuoles containing granular eosinophilic material within it (►Fig.3).

Discussion
Our patient had onset of symptoms in the second decade with features of indolent proximal lower limb involvement and imaging features suggesting preferential involvement of adductor component of thigh muscles and also classical histopathological findings with next-generation sequencing confirming the diagnosis.][9][10][11][12][13][14][15][16][17] XMEA affects males and has a slowly progressive proximal limb muscle weakness and normal cardiac function with onset ranging from childhood to adulthood. 10VMA21 is an assembly chaperone for the principal mammalian proton pump required for lysosome acidification.Due to the genetic defect in VMA21, alteration in lysosomal acidification leads VMA21-Associated Myopathy with Excessive Autophagy Bardhan et al.
to a block in steps of macroautophagy and accumulation of autophagosomes.This results in vacuole formation filled with debris in the muscles. 2 These vacuolar changes can also be observed in Danon's disease; however, calcium deposition, basal lamina reduplication, and absence of cardiomyopathy suggest XMEA. 1,18ost VMA21 mutations have been reported to affect normal splicing, predominantly occurring in the introns 1 and 2. One missense, c.272G > C, and another synonymous mutation, c.294C > T, while present in exon 3 have also been shown to affect splicing ( 12,13 ).Mis-splicing mutations lead to the reduction of mRNA and, in turn, reduced expression of VMA21 protein. 2 Clinical phenotype severity has been described as directly related to the extent of mRNA expression reduction. 12,13Intronic VMA21 mutations affecting the polypyrimidine tract in intron 2 adjacent to the acceptor site (c.164-6T > G; c.164-7T > G; c.164-20T > A) have been reported in multiple patients. 3,5,16The mutations -6T > G and -20T > A pyrimidine-to-purine substitutions have been associated with severe phenotypes of congenital onset CAVM and childhood onset with relatively severe progression, respectively. 5,12In comparison, c.164-7T > G has been reported with milder XMEA phenotype with variable onset ranging from childhood to late adulthood (>50 years). 3,16The pyrimidine-to-purine substitution at -7 position in intron 2 has been described to reduce the binding efficacy of the U2AF splice factor, causing abnormal splice recognition at the acceptor site. 2 Our patient also had a novel pyrimidine-to-purine change of T > A at the same -7 position reported in earlier XMEA patients.While we could not check the mRNA expression levels in our patient, based on in-silico predictions, we presume a similar impact as in previous patients with -7T > G substitution.This correlated with our patient's milder XMEA phenotype with second-decade onset.F-3: proximal lower limb weakness.
F-4: persistently elevated CK.F-5: infantile hypotonia followed by proximal lower limb weakness with a rigid spine.
F-1:  VMA21-Associated Myopathy with Excessive Autophagy Bardhan et al.The age of onset is quite variable, though typically described as childhood onset.There are various reports on adult-onset myopathy, as noted in our patient, 4,9,15 with the oldest being onset in the seventh decade, as reported by Crockett et al. 3 It typically affects males and spares carrier females.The phenotypic spectrum extends from floppy infant delayed milestones to proximal limb-girdle and respiratory muscle weakness.Usually, extra skeletal muscle involvement does not occur.However, Fernandes et al described a patient with childhoodonset distal lower limb weakness with incomplete right bundle branch block. 17he earliest and most frequently affected muscles in XMEA are the pelvic girdle and proximal thigh muscles, with the anterior compartment more involved than the posterior compartment, mimicking limb girdle muscular dystrophy pattern.MRI pattern of muscle in XMEA typically shows early involvement of the vasti, adductor magnus, and soleus with relative sparing of the rectus femoris and tibialis anterior. 5Although muscle imaging shows thigh involvement in almost all patients, few patients show preferential involvement of the adductor group. 1,16However, selective involvement of peripheral vastus lateralis, partial rectus femoris, and peripheral tibialis anterior muscle has also recently been described. 11ur patient also had typical findings as described in previous reports with fatty infiltration of the vasti and adductor magnus and sparing of rectus femoris, showing that imaging pattern could be a strong indicator toward XMEA diagnosis in clinically suspected patients.
Histopathological analysis of skeletal muscle may show muscle fibers with sarcoplasmic vacuoles containing degraded organelles, debris, and calcium crystals.These vacuoles can be seen within the muscle fibers' interior depths or superficially close to the sarcolemma. 18Generally, inflammation, necrosis, or apoptosis are not observed.Instead, myofiber demise occurs through a novel form of autophagic cell death characterized by giant autophagic vacuoles, 2 to 10 µm in size, encircling sections of cytoplasm, including organelles.These vacuoles contain lysosomal hydrolases with incomplete contents of digestion.Definitive diagnosis includes clinical presentation and genetic mutation identification.

Conclusion
Here, we report an Indian case of VMA21-related myopathy that adds to our knowledge of XMEA phenotype-genotype spectrum.The pathological features and clinical spectrum of XMEA are large and overlap with other disorders.It also highlights the preferential involvement of thigh muscles with distinctive sparing of rectus femoris, which may aid in the diagnosis of this rare myopathy.

Prior Publication
None.

Fig. 1
Fig. 1 Pedigree chart of the patient.

Fig. 2
Fig.2Muscle MRI of the patient.(A) T1W, (B) T2W, and (C) STIR sequences axial sections in the midthigh region demonstrate fatty atrophy and volume loss of bilateral vastus medialis, intermedius, and lateralis with involvement of adductor magnus.Rectus femoris is relatively spared (arrow).In the bilateral thigh muscles, there was no aberrant STIR signal intensity.

Fig. 3
Fig. 3 Histopathology of muscle biopsy.(A,B) Microphotograph showing a transverse section of skeletal muscle with variation in fiber size including atrophic angulated fibers (asterisk Ã ), internalized nuclei (^) and a few hypertrophic fibers.(H&E stain; scale bar A, B-100 µm) Inset (i) shows a vacuole in one of the myofibers (H & E, scale bar-20 µm).(C-F) Microphotograph showing a transverse section of skeletal muscle with variation in fiber size including atrophic angulated fibers (asterisk, Ã ) and a few hypertrophic fibers.A few of the fibers show vacuoles with granular eosinophilic material within (arrows).Internalized nuclei (^) and a regenerating fiber (r) are observed.Occasional fiber shows splitting (!).Also note the interstitial/endomysial fibrosis as evident in E and F. (Fig C-H & E, scale bar 50 µ; D, E, F-Masson's trichrome stain, scale bar: D-50 µm; E, F-20 µm).

Table 1
Comprehensive details of salient features of current patient and published studies Global Medical Genetics Vol.11 No. 2/2024 © 2024.The Author(s).VMA21-Associated Myopathy with Excessive Autophagy Bardhan et al. 171